Infections of humans with human cytomegalovirus (HCMV) are widespread and as a rule proceed without symptoms during childhood. HCMV-specific IgG antibodies, as markers for an infection which has taken place or is latent, can be detected in 50-90% of adults, depending on the population tested. Congenital infection and acute infections in immunosuppressed patients, such as transplant patients and HIV-infected patients, can lead to a life-threatening disease.
To ensure early and effective antiviral treatment, it is of great interest to establish whether an acute infection is present. Various methods can be used for diagnosis of an acute HCMV infection. In transplant patients in particular, in whom constant, concomitant diagnostics are possible, the 'pp65-specific antigenemia test and the polymerase chain reactions, or PCR have become largely accepted in recent years because of their superior sensitivity. In the routine laboratory, an acute infection is as a rule diagnosed, for example in the context of viral differential diagnosis or during pregnancy screening, by detection of the pathogen in the cell culture and serologically by the detection of specific antibodies, in particular in an enzyme linked immunosorbent assay or ELISA. More or less purified, poorly characterized lysates of HCMV-infected cell cultures are currently exclusively used as specific antigens for the antibody detection. This leads to specificity and sensitivity problems, especially in IgM detection. Numerous publications exist which deal with the diagnostic relevance of various recombinant antigens and chemically synthesized peptides. The results available to date are summarized in M. P. Landini: "Antibody Responses to Human Cytomegalovirus Proteins", Reviews in Medical Virology 2, 63-72 (1992) incorporated by reference.
The object of the present invention is therefore to provide polypeptides and/or fusion proteins which are advantageous for diagnostics, as well as test kits which contain these and methods for the detection of acute infections of HCMV.
In the context of the present application, the term polypeptides is understood as meaning biomolecules which consist of .alpha.-amino acids and which contain more than 10 amino acids. In respect of the upper limit of the polypeptides, it is to be noted that these are not complete proteins or virtually complete proteins. If the polypeptides according to the invention are in the form of fusion proteins, it is also entirely possible for them to contain more than 100 amino acids. If they are in the form of fusion proteins, it is entirely possible for the polypeptides according to the invention to have a length of up to about 300 amino acids. It is essential, however, that the polypeptides according to the invention contain only certain regions from the proteins of cytomegalovirus.
For diagnostic tests, two aspects are of particular importance. First, the antigens must be highly specific, i.e. in the diagnostic test it must be clearly recognizable that the pathogen is that sought and not a different pathogen. Second, the diagnostic test must be highly sensitive, i.e. every infection with the pathogen sought must be detectable as reliably as possible even if only relatively few antibodies are present in the sample to be analyzed. As far as possible, no false positive results should occur.
The individual proteins encoded by the human cytomegalovirus are as a rule characterized by the reading frame by which they are coded. Landini (loc. cit.) has reported, for example, the genomic location of the various proteins already known for HCM virus. The reading frame can therefore be used for characterization of the protein.
The reading frame UL57 of the human cytomegalovirus codes for the so-called major DNA-binding protein, which has a molecular weight of about 130 to 133 kD. Anders et al. characterized the protein and the reading frame in J. Virology, 62, pages 1364-1372 (1988). Interestingly, the reading frame UL57 has significant homology to the corresponding reading frames of other herpesviruses, in particular to the viruses HHV-6 and to a lesser extent to EBV (Epstein-Barr virus). On the basis of this homology to the reading frames of other viruses, the major DNA-binding protein encoded by UL57 would not actually appear suitable for diagnostics since there is the risk of cross-reactions.
However, in the context of the present invention it has been found, surprisingly, that regions from the reading frame UL57 can be used in a particularly advantageous manner for diagnostics, since polypeptides and fusion proteins which contain this amino acid sequence allow advantageous results to be obtained in diagnostic tests. In the context of the present invention, three fragments were provided from the reading frame UL57, where it was possible for the two fragments UL57/1 and UL57/3 to be amplified by means of the polymerase chain reaction and the fragment UL57/2 was provided by means of chemical DNA synthesis. These DNA fragments were recloned in an expression vector (pGEX-3) which allows expression of the viral proteins in the form of fusion proteins (with glutathione S-transferase (GST)). The fusion proteins were purified and tested with selected sera in ELISA experiments. In these, it was found, surprisingly, that the antigenic fragment UL57/3 consisting of 57 AA is an outstanding antigen for specific and sensitive detection of IgM antibodies present during acute HCMV infection. In contrast, the other two fragments proved to be not diagnostically relevant. Since UL57/3 can be expressed as a non-fusion protein in E. coli only with difficulty, because of its small size, expression was also carried out as an autologous fusion protein together with other diagnostically relevant HCMV antigen fragments, which likewise ensure specific and sensitive IgM serodiagnostics. In the case of the autologous fusion protein 52/3 57/3, expression was carried out C-terminally with amino acids 297-433 of p52. In the case of autologous fusion protein 52/3 57/3 150/7, the 54 C-terminal amino acids of pp150 were additionally expressed at the C-terminal end of 57/3. UL57/3 was furthermore synthesized chemically as a peptide.
The present invention therefore relates to polypeptides comprising an amino acid sequence originating from cytomegalovirus (HCMV), which originates from the reading frame UL57 of the HCM-virus, this reading frame coding for the major DNA-binding protein, which have a homology of at least 60% to the amino acid sequence (SEQ ID NO: 1):
Gly Val Pro Gly Gly Gly Ala Gly Gly Gly Gly Gly Arg Asp Val Ser Gly Gly Pro Ser Asp Gly Leu Gly Gly Gly Arg Gly Gly Gly Gly Gly Gly Asp Ser Gly Gly Met Met Gly Arg Gly Gly Arg Met Leu Gly Ala Ser Val Asp Arg Thr Tyr Arg Leu Asn
The polypeptides according to the invention preferably have a homology of at least 80% to the amino acid sequence given above. In a particularly preferred embodiment, the polypeptides according to the invention have the amino acid sequence given above or a part sequence thereof. The polypeptides according to the invention usually have a length of at least 10 amino acids; however, polypeptides with a length of at least 25 amino acids are preferred, and polypeptides with a length of at least 40 amino acids are particularly preferred.
The peptides according to the invention can be synthesized by chemical methods which are known per se (for example solid phase synthesis), or can also be prepared by genetic engineering methods.
EPA 87.111726.3 describes a structural phosphoprotein (pp150) of human cytomegalovirus which has proved to be important for detection of specific antibodies.
IgG antibodies directed against HCMV show that infection with human cytomegalovirus has taken place at some time. On the other hand, by determining the titer of IgM antibodies against HCMV, it can be established whether a fresh infection with HCM-viruses is present or not. The answer to this question is of great importance especially the high risk patients mentioned supra for pregnant women. It is of particular importance with such a test that the detection is highly specific and highly sensitive. If too high a number of positive but non-confirmable results is obtained in the test, the test has only a low conclusiveness. On the other hand, however, the test must also reveal fresh HCMV infections with sufficient certainty, but the number of falsely positive results should be reduced as far as possible.
The DNA sequence of cytomegalovirus is already known. Chee et al. disclose, in their publication in Current Topics in Microbiology and Immunobiology, Vol. 154 (1990), pages 125-169, incorporated by reference the locations of the DNA sequence and an analysis of the coding sequences of cytomegalovirus.
In the context of the present invention, it was found that the C-terminal region of the pp150 tegument protein is a very sensitive antigen for detection of IgM antibodies. In the context of the present invention, this is a region of 40 to 60 amino acids on the C-terminus of the pp150 protein. Since the amino acid sequence of pp150 protein is already known, this is the region of amino acids 988 to 1048. The region of the pp150 tegument protein which comprises amino acids 994 to 1048 is particularly preferred in the context of the present invention, and this region is presently designated as pp150/7/2.
According to the invention, this C-terminal region of the pp150 tegument protein binds to a region of high IgM reactivity which originates from another antigen of the HCM-virus. Other antigens are, for example, the antigens called pp65, pp71, pp28 or gp116/58. The antigen p52, and in particular the C-terminal region of the antigen p52, has proven to be a particularly suitable antigen in the context of the present invention.
In the context of the present invention, a region from the C-terminus of p52 which contains about 100 to 150 amino acids of the p52 antigen is thus preferably employed as the other component of the fusion protein. The region from p52 which comprises amino acids 297 to 433 is especially preferred in the context of the present invention.
In the context of the present invention, it has been found that the tegument protein pp150 showed a very high IgM reactivity with sera from healthy blood donors, whereby the value of this antigen is significantly reduced for detection of acute infections. A proportion of about 35% of positive results in the IgM test in healthy, seropositive blood donors is not realistic and shows that the whole pp150 antigen is unsuitable for detection of the IgM antibodies.
It has furthermore been found that the C-terminal region of the p52 antigen merely shows a positive reaction with a serum. Thus, if only p52 were to be used as an antigen, the sensitivity of the assay would not be adequate. The present invention thus also relates to fusion proteins which are prepared by recombination and contain at least one fragment from the C-terminal region of the tegument protein p150 of cytomegalovirus (HCMV) and at least one further fragment from another antigenic protein originating from human cytomegalovirus.
In a preferred embodiment, the other antigenic protein of the fusion protein originates from the protein called p52, wherein a particularly preferred embodiment uses the p52 C-terminal region. In an especially preferred embodiment, this region extends from about the amino acid in position 283 to amino acid No. 433 of p52.
The amino acid sequence of a particularly preferred fusion protein is shown in FIG. 2. For immunological tests, the amino acids can of course be exchanged for amino acids which have functionally the same action without the action of the fusion protein being changed. The expert is fully aware that amino acid exchanges do not affect the immunological properties of a protein as long as the essential structure of the epitopes is not changed. The invention therefore also relates to those fusion proteins which have a homology of at least 80%, and in a particularly preferred form of at least 90%, to the fusion protein shown in FIG. 2. A homology of 80% means that 80 out of 100 amino acids at corresponding points are identical, while 20% of the amino acids may vary.
The fusion proteins just described, which are used according to the invention, comprise (i) the C-terminal part of pp150 and (ii) a region of high IgM reactivity of another cytomegalovirus antigen. Furthermore, the fusion proteins according to the invention can also contain a short amino acid sequence due to the recombinant preparation of the fusion protein. According to the invention, in a preferred embodiment, however, this amino acid sequence, which originates in particular from the expression vector, contains not more than 25 amino acids.
The fusion proteins which can be prepared in this way have an amino acid sequence of a polypeptide or of a fusion protein according to one of claims 1 to 15. Some of the fusion protein preferably originates from glutathione S-transferase. The fusion proteins according to the invention can contain either a polypeptide from the reading frame UL57 of HCM-virus, or a portion of at least one other antigenic protein of human cytomegalovirus. In a particularly preferred embodiment, the other antigenic protein of human cytomegalovirus is the pp150 tegument protein or the p52 protein. Combinations of part sequences of the abovementioned proteins are particularly preferred.
The present invention also relates to test kits for the detection of antibodies, in particular IgM antibodies, against cytomegalovirus, which comprise at least one polypeptide according to the invention and/or one fusion protein according to the invention. These are preferably test kits for carrying out an ELISA test.
Several different test methods, such as Western blot, radio-immuno-assay and others, are known in diagnostics. Test kits which are particularly preferred in the context of the present invention are those which are suitable for carrying out the ELISA test (enzyme-linked immunosorbent assay). Coupling to a solid phase takes place in these tests. The antigen, in the present case the fusion protein, is often bound to a solid phase, in particular on microtiter plates, and nonspecific bindings sites are saturated. The sample to be analyzed, usually serum, is then introduced into these microtiter plates, incubated and washed. The antibodies bound to the solid phase are then directed against the antigen to be detected. These antibodies are as a rule detected with anti-human antibodies, to which an indicator component is bound. Horseradish peroxidase, which catalyzes a color reaction with the aid of which the test result can be read off, has proved to be particularly suitable here.
An alternative method for detection of specific IgM antibodies is .mu.-capture ELISA. In this method, the solid phase of a carrier, for example an ELISA plate, is coated with polyclonal or monoclonal antibodies directed against the .mu. chain of the human IgM molecule. On subsequent incubation with human serum, IgM antibodies bind selectively to the solid phase, while antibodies of all other classes of antibodies are removed by washing. The CMV-specific IgM antibodies are determined from the large number of bound IgM molecules by means of an enzyme-labeled CMV antigen which is bound by the IgM molecules directed against this antigen. The antibody-antigen complex formed can be detected by an enzyme-mediated color reaction--as already described above.
The present invention also relates to methods for the detection of antibodies against HCMV, in which the sample to be analyzed is brought together with a fusion protein. The antibody-polypeptide or fusion protein complex can then be detected with a detection component. Antigens which display at least some of the "additional sensitivity" of pp150, but at the same time have a high specificity, are also provided by the polypeptides and fusion proteins according to the invention. Only a low IgM reactivity with sera of healthy blood donors is therefore obtained with the polypeptides and fusion proteins according to the invention. With the aid of the polypeptides and fusion proteins according to the invention, it is thus possible to carry out a test for IgM antibodies which detects with adequate sensitivity whether an acute infection is present, and which at the same time considerably reduces, if not excludes completely, the occurrence of false positive results.
Antigens which have a high sensitivity but at the same time a high specificity are provided by the fusion proteins and peptides according to the invention. Only low IgM reactivity with sera of healthy blood donors is therefore obtained with the peptides and fusion proteins according to the invention. With the aid of the peptides and fusion proteins according to the invention, it is thus possible to carry out a test for IgM antibodies which detects with adequate sensitivity whether an acute infection is present and which at the same time considerably reduces, if not even excludes, the occurrence of results which are not clinically relevant or are even false positives.
The detection component is preferably an anti-human antibody which is coupled to an indicator molecule, in particular horseradish peroxidase.
The disclosure of the present invention also enables the expert to synthesize oligonucleotides which comprise a sequence of oligonucleotides which code for an amino acid sequence which corresponds to at least part of the amino acid sequence given in claim 1. The oligonucleotides are preferably 12-30 bases long. They are usually DNA oligonucleotides which can be used for the polymerase chain reaction or other processes which allow amplification of nucleic acids.
The present invention also relates to oligonucleotides which comprise a nucleotide sequence which codes for at least some of the polypeptide UL57/3 according to the invention. Such oligonucleotides can advantageously be employed as a primer in the polymerase chain reaction. Sensitive detection of cytomegalovirus is possible with the aid of the polymerase chain reaction. With this generally known method, detection is possible, e.g., in cases where only a small number of copies of the genetic information of the virus is present.
The term "homology" is understood as meaning the degree of agreement of two DNA or amino acid sequences. 60% homology means, for example, that 60 out of 100 amino acid positions in the sequences coincide. The homology of proteins is determined by sequence analysis.
Since human cytomegalovirus has already been researched relatively thoroughly, the sequence and the reading frame which comprises the peptide according to the invention can be determined with the aid of the publication by Chee et al., Current Topics in Microbiology and Immunology, 154, Springer Verlag (1990), pages 126-169 incorporated by reference.
Fusion proteins which are preferred according to the invention have on the one hand an amino acid sequence of the polypeptide UL57/3 according to the invention and on the other hand a fragment of another protein, in particular another antigenic protein, of human cytomegalovirus and/or a fragment originating from the expression vector. These further constituents of the fusion proteins particularly preferably originate from the tegument protein pp150 and/or from the protein p52 of cytomegalovirus. In an especially preferred form, these are the fragments which are described in the examples of this application.
In the context of the present invention, it has also been found that those fusion proteins which contain at least a portion of glutathione S-transferase (GST) can be expressed easily in E. coli. The vectors with which such fusion proteins can be provided has been described, in particular, by Vornhagen et al., Journal of Clinical Microbiology, 32, pages 981-986 (1994).
In the context of the present invention, the antigens were evaluated with:
a) nonselected sera of healthy blood donors without signs of acute HCMV infection. The sera were classified into HCM-positive/-negative with an anti-CMV IgG ELISA approved by the Paul Ehrlich Institute (Biotest), (See Table 5, infra). PA1 b) selected sera of immunocompetent (functioning immune system) individuals with an acute HCMV infection, documented by a positive result in virus isolation and/or by IgG seroconversion (See Table 3, infra). PA1 c) selected sera of transplant patients with an acute HCMV primary infection, (See Table 4, infra). The acute infection was detected with the aid of the pp65-specific antigenemia test.